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Abstract

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops
cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth
factor (HGF) is an important mitogen and morphogen that contributes to the repair
process after tissue injury, we investigated the role of HGF in cutaneous fibrosis
and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected
in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes
containing 8 μg of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector
(untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects
of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-β
and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production
by TSK/+ CD4+ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness,
including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated
TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice.
However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-β1 mRNA in
the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture
and examined the effect of recombinant HGF on the generation of IL-4. Recombinant
HGF significantly inhibited IL-4 production in TSK/+ CD4+ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-β mRNA expression, which has been postulated
to have a major role in fibrinogenesis and reduced hypodermal thickness, including
the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel
strategy for the treatment of SSc.

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder of unknown etiology that
is characterized by an excessive deposition of extracellular matrix protein in the
affected skin, in addition to various internal organs. Currently, no effective therapies
for SSc exist [1]. The tight-skin (TSK/+) mouse is a genetic model for human SSc. Although the phenotypic
characteristics of the TSK/+ mouse are not identical to those of human SSc patients,
TSK/+ mice produce autoantibodies against SSc-specific autoantigens, including topo-I,
fibrillin 1 (fbn-1), collagen type 1, and Fcγ receptors [2,3]. The gene defect responsible for the TSK/+ phenotype in mice is yet to be definitively
identified; however, the fbn-1 gene has been recently proposed as a candidate locus for this disorder [4]. The fbn-1 gene mutation seems to provide an explanation for the embryonic lethality of homozygous
tight skin (TSK/TSK) animals. Heterozygous (TSK/+) mice, which have a normal lifespan,
exhibit fibrosis and thickening of subcutaneous dermal tissue. Other abnormalities
in TSK/+ mice include increased lung collagen content, enlarged air spaces reminiscent
of pulmonary emphysema, and, with advanced age, development of progressive myocardial
fibrosis and hypertrophy [5-7].

Hepatocyte growth factor (HGF) was originally identified and cloned as a potent mitogen
for hepatocytes [8,9]. It has mitogenic, motogenic, and morphogenic effects on various epithelial tissues,
including the liver, kidneys, lungs, and intestines [10-12]. HGF also shows antiapoptotic activity [13] and has a role in suppressing fibrosis in the liver [14]. HGF might, therefore, induce tissue repair in dermal sclerosis associated with SSc.
We recently demonstrated that repeated transfection of the human HGF gene into skeletal muscle induced continuous production of HGF, strongly inhibited
acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem-cell transplantation
(HSCT), and protected against thymic damage caused by GVHD in a well-characterized
mouse model of GVHD [15,16]. The present study was performed to examine the therapeutic effect of HGF on tissue
fibrosis in TSK/+ mice.

Materials and methods

Animals

TSK/+ and homozygous pallid (pa/pa) mutant mice with a C57BL/6 background were obtained
from the Jackson Laboratory (Bar Harbor, ME, USA). TSK/+ mice are heterozygous for
both TSK and pa gene mutations. TSK/+ mice were produced by mating TSK/+ male mice with pa/pa female
mice. TSK/+ progeny were identified by their back-coat and eye colors, in addition
to their characteristic loss of skin pliability. To verify the TSK/+ genotype, PCR
amplification of a partially duplicated fbn-1 gene was carried out using genomic DNA from each mouse, as described previously [17]. C57BL/6 and (C57BL/6 × DBA/2)F1 (BDF1) mice were obtained from the Shizuoka Laboratory
Animal Center (Hamamatsu, Shizuoka, Japan). All mice were maintained in a pathogen-free
facility at the Hyogo College of Medicine (Nishinomiya, Hyogo, Japan). Animal experiments
were performed in accordance with the guidelines of the National Institutes of Health
(Bethesda, MD, USA), as specified by the animal care policy of Hyogo College of Medicine.

Human HGF cDNA (2.2 kb) was inserted into the EcoRI and NotI sites of the pUC-SRα
plasmid under control of the cytomegalovirus enhancer-promoter [18]. HVJ liposomes containing plasmid DNA and high mobility group 1 protein were constructed,
as described previously [15,16]. Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed at
a weight ratio of 1 : 4.8 : 2. This lipid mixture (1 mg) plus plasmid DNA (20–40 μg),
which had previously been complexed with 6–12 μg of high mobility group 1 nonhistone
chromosomal protein purified from calf thymus, were sonicated to form liposomes and
then mixed with ultraviolet-irradiated HVJ. Excess free virus was subsequently removed
from the HVJ liposomes by sucrose-density-gradient centrifugation.

Histopathology

Tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections were
stained with hematoxylin and eosin and were examined by light microscopy.

RT-PCR

RNA was extracted using an Isogen (Nippongene, Toyama, Japan) kit, according to the
manufacturer's instructions, and cDNA was prepared using 2.5 μM random hexamers (Applied
Biosystems Inc., Foster City, CA, USA). IL-4 and TGF-β mRNA levels were quantified
by real-time RT-PCR in a total volume of 25 μl for 40–50 cycles of 15 seconds at 95°C
and 40–50 cycles of 1 minute at 60°C. Samples were run in triplicate and relative
expression levels were determined by normalizing expression levels to that of β-actin.
The primer sequences used were as follows:

ELISA for IL-4

The levels of murine IL-4 in culture supernatants were measured by ELISA using antimouse
IL-4 mAb (Genzyme Pharmaceuticals, Cambridge, MA, USA) according to the manufacturer's
protocol.

Statistical analysis

Group mean values were compared by the two-tailed Student's t-test. A p value of < 0.05 was considered statistically significant.

Results

Prevention of scleroderma by HGF gene transfection

We previously reported that repeated transfection of the human HGF gene into skeletal muscle of allogeneic HSCT recipients reduced the tissue damage
and subsequent inflammatory responses caused by acute GVHD [15,16]. To investigate the possible therapeutic effects of HGF on SSc, young TSK/+ mice
were treated with HGF gene transfection. Treatment consisted of once-weekly intramuscular injection of either
HGF-HVJ liposomes or control mock vectors for a period of 8 weeks (Figure 1). Histologic analysis revealed that HGF treatment of TSK/+ mice resulted in a marked
reduction of hypodermal thickness, including the subcutaneous connective tissue layer
(Figure 2). Skin fibrosis was assessed quantitatively by measuring hypodermal thickness. The
hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold
compared with untreated TSK/+ mice (Figure 3).

Figure 1. Experimental protocol for injection of HGF-HVJ liposomes into TSK/+ mice. Treatment
consisted of once-weekly injection of either HGF-HVJ liposomes or control mock vector
for a period of 8 weeks. Treatment was initiated at the age of 4 weeks. Histopathology
and cytokine mRNA expression in the spleen, skin, and lungs was examined 8 weeks after
treatment. HGF, hepatocyte growth factor; HVJ, hemagglutinating virus of Japan; im,
intramuscular; TSK/+, tight skin.

Effect of HGF on the expression of IL-4 and TGF-β mRNA expression and production

IL-4 and TGF-β have been postulated to have major roles in fibrinogenesis in animal
models [19-23]. To clarify whether modulation of IL-4 and TGF-β has a role in the prevention of
sclerosis induced by HGF gene transfection in the scleroderma model mouse, we examined the mRNA expression
of these cytokines. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-β1 mRNA in
the spleen and skin but not in the lung (Figure 5). We also performed MLR and examined the effect of HGF on the production of IL-4.
Responder CD4+ T cells from TSK/+ mice were cultured with irradiated (20 Gy) CD11c+ DCs from BDF1 mice with or without recombinant HGF. After 3 days' culture, viable
cells were stimulated by culture with anti-CD3 mAb for 48 hours and the IL-4 level
in the culture supernatant was assayed by ELISA. HGF significantly inhibited IL-4
production from TSK/+ CD4+ T cells stimulated by BDF1 DCs (Figure 6).

Discussion

SSc is an autoimmune connective tissue disease that is characterized by microvascular
damage, extracellular matrix deposition, and fibrosis. There is no completely effective
treatment for this disease at present. We previously demonstrated that serum HGF levels
were significantly elevated in patients with SSc and serum HGF levels correlated to
markers of endothelial injury (thrombomodulin) and interstitial lung injury (KL-6),
suggesting that elevation of serum HGF counteracts the endothelial and interstitial
lung injury caused by SSc [24]. The serum level of HGF is significantly elevated in various diseases, depending
on the severity of the disease [25-27]. However, endogenously induced HGF is not sufficient to repair tissue injuries, and,
therefore, supplementation with exogenous HGF is necessary to accelerate the tissue
repair process in animal models [14,15,28]. In the present study, we assessed the effect of exogenous HGF on skin fibrosis and
the development of pulmonary defects in the TSK/+ mouse model of SSc. Both our present
study and other previous studies [5] have shown that dermal thickness is similar in TSK/+ and wild-type littermates, but
hypodermal thickness, including the subcutaneous connective tissue layer, is significantly
increased in TSK/+ mice compared with wild-type littermates. HGF gene transfection of TSK/+ mice for a period of 8 weeks resulted in a marked reduction
of hypodermal thickness, including the subcutaneous connective tissue layer. Although
the therapeutic effect of HGF is not significant, we also observed the reduction of
hypodermal thickness in TSK/+ mice following HGF gene transfection for a period of 4 weeks (data not shown).

Although the cause of SSc is unknown, IL-4 and TGF-β have been postulated to have
major roles in fibrinogenesis. In one study, intravenously administered human immunoglobulin
decreased splenocyte secretion of IL-4 and TGF-β, which resulted in abrogation of
fibrinogenesis in TSK/+ mice and, consequently, prevented the accumulation of fibrous
tissue [19]. Furthermore, administration of an anti-IL-4 or anti-TGF-β antibody prevented dermal
collagen deposition in TSK/+ mice and murine sclerodermatous GVHD, respectively [20,21]. In the present study, HGF treatment also reduced expression of both IL-4 and TGF-β
mRNA in the spleen and skin.

IL-4 regulates collagen and extracellular matrix production by fibroblasts [22,23]. TSK/+ mice exhibiting disrupted genes encoding IL-4 receptor alpha (IL-4Rα) or IL-4
lacked skin sclerosis [17,29], suggesting that IL-4 has a crucial role in skin sclerosis in TSK/+ mice. A primary
source of IL-4 in vivo is CD4+ T cells [30] and a previous study demonstrated that CD4+ T cells were essential to the TSK/+ phenotype, because a lack of these cells prevented
development of dermal thickening [31]. Therefore, we examined the effect of HGF on the generation of IL-4 from CD4+ T cells. HGF significantly inhibited IL-4 production from CD4+ T cells stimulated by allogeneic DCs, suggesting that HGF inhibits dermal fibrosis,
in part, by inhibiting IL-4 production by CD4+ T cells.

We also observed downregulation of TGF-β1 mRNA expression in TSK/+ mice by HGF gene transfection. TGF-β1 has a role in the induction of fibrosis, and HGF gene transfection inhibited the production of TGF-β1 from macrophage-like cells and
fibroblastic cells [32]. Downregulation of TGF-β1 expression and inhibition of fibrosis by HGF were noted
in a rat model of liver cirrhosis [14] and a mouse model of chronic renal failure [33]. Recently, HGF has been shown to downregulate TGF-β1 expression and prevent dermal
sclerosis in a murine bleomycin-induced scleroderma model [34]. The authors observed that HGF gene transfection significantly reduced both the expression of TGF-β1 mRNA and the
production of TGF-β1 by fibroblastic cells and macrophage-like cells that infiltrated
the dermis. Furthermore, HGF gene transfection prevented the symptoms of not only dermal sclerosis, but also lung
fibrosis induced by bleomycin injection.

By contrast, HGF gene transfection failed to alter the development of pulmonary abnormalities in TSK/+
mice in our study. The pathologic alteration of the lung structure of TSK/+ mice represents
pulmonary emphysema and is not related to hypersynthesis of collagen that is similar
to the pulmonary fibrosis associated with SSc [35]. Apparently, emphysema in TSK/+ mice is not owing to the mutated fbn-1 gene that is responsible for the occurrence of cutaneous hyperplasia, because transgenic
mice bearing a mutated fbn-1 gene developed cutaneous hyperplasia but did not exhibit pulmonary emphysema [36]. Furthermore, TSK/+ mice exhibiting disrupted genes encoding IL-4Rα, TGF-β, or IL-4
lacked skin sclerosis but developed emphysema, indicating that different genes are
involved in the development of skin sclerosis and pulmonary emphysema in TSK/+ mice
[17,29]. Furthermore, other studies have shown that the pulmonary pathology remained unchanged
in TSK/+ CD4 knockout (CD4-/-) mice [31] and TSK/+ mice treated with an anti-IL-4 antibody [20]. The dermal and pulmonary components of the TSK/+ phenotype can, therefore, be dissociated
in vivo.

We used a transgenic approach instead of using a recombinant protein for the following
reasons:

1. Because the half-life of a recombinant protein is quite short, recombinant protein
treatment needs an enormous dose of the active form of HGF protein and frequent injections.

2. Administering a high dose of the active form of the HGF protein could cause adverse
effects, such as tumorigenesis in other organs [37].

3. Recombinant protein treatment is very costly.

By contrast, the transgenic approach is simple, safe, and cheap and needs much less
frequent injections. Repeated weekly injection of HGF-HVJ liposomes achieves a continuous
high plasma level of HGF [14-16].

Although further studies are needed to fully explore the effects of HGF on SSc, it
is possible that HGF therapy might be a promising strategy for the treatment of SSc.

Conclusion

HGF gene transfection of TSK/+ mice resulted in a marked reduction of hypodermal thickness,
including the subcutaneous connective tissue layer. However, TSK/+-associated defects
in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-β1 mRNA in
the spleen and skin but not in the lung. Recombinant HGF significantly inhibited IL-4
production by TSK/+ CD4+ T cells stimulated by allogeneic DCs. HGF might represent a novel strategy for the
treatment of SSc.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

T Iwasaki conceived the study, participated in the design and co-ordination of the
study, and participated in the interpretation of the results. T Imado and SK performed
the animal study and histologic analysis. HS participated in the design of the animal
study.

Acknowledgements

T Iwasaki acknowledges the support of a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science and Culture of Japan (No. 15591071).

References

LeRoy EC: A brief overview of the pathogenesis of scleroderma (systemic sclerosis).